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Post a Comment 1 Sector of Organic Chemistry and Biochemistry, University of Ioannina, Ioannina, Greece
2 DOE Joint Genome Institute, Walnut Creek, California, USA
3 Los Alamos National Laboratory, Bioscience Division, Los Alamos, New Mexico, USA
4 Biological Data Management and Technology Center, Lawrence Berkeley National Laboratory, Berkeley, California, USA
5 Department of Cell Biology and Biophysics, Faculty of Biology, University of Athens, Athens, Greece
6 Centre for Bioinformatics - Department of Informatics - School of Natural & Mathematical Sciences, King's College London (KCL) - London, UK
7 Present address: Computational Genomics Unit, Institute of Agrobiotechnology - Centre for Research & Technology Hellas - Thessaloniki - Greece
Electronic publication date: April 29, 2011.
Abstract
Arthrobacter phenanthrenivorans is the type species of the genus, and is able to metabolize phenanthrene as a sole source of carbon and energy. A. phenanthrenivorans is an aerobic, non-motile, and Gram-positive bacterium, exhibiting a rod-coccus growth cycle which was originally isolated from a creosote polluted site in Epirus, Greece. Here we describe the features of this organism, together with the complete genome sequence, and annotation.
Keywords: Arthrobacter, dioxygenases, PAH biodegradation, phenanthrene degradation.
Kallimanis et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Strain Sphe3T (=DSM 18606T = LMG 23796T) is the type strain of Arthrobacter phenanthrenivorans [1]. It was isolated from Perivleptos, a creosote polluted site in Epirus, Greece (12 Km North of the city of Ioannina), where a wood preserving industry was operating for over 30 years [2]. Strain Sphe3T is of particular interest because it is able to metabolize phenanthrene at concentrations of up to 400 mg/L as a sole source of carbon and energy, at rates faster than those reported for other Arthrobacter species [3-5]. It appears to internalize phenanthrene with two mechanisms: a passive diffusion when cells are grown on glucose, and an inducible active transport system, when cells are grown on phenanthrene as a sole carbon source [2]. Here we present a summary classification and a set of features for A. phenanthrenivorans strain Sphe3T, together with the description of the complete genome sequencing and annotation.
Figure 1 shows the phylogenetic neighborhood of A. phenanthrenivorans strain Sphe3T in a 16S rRNA based tree.
Figure 1 Phylogenetic tree highlighting the position of A. phenanthrenivorans strain Sphe3T relative to the other type strains within the family. Numbers above branches are support values from 100 bootstrap replicates. |
Strain Sphe3T is a Gram-positive, aerobic, non-motile bacterium exhibiting a rod-coccus cycle (Figure 2), with a cell size of approximately 1.0-1.5 x 2.5-4.0 μm. Colonies were slightly yellowish on Luria agar. The temperature range was 40-37oC with optimum growth at 30-37oC. The pH range was 6.5-8.5 with optimal growth at pH 7.0-7.5 (Table 1). Strain Sphe3T was found to be sensitive to various antibiotics, the minimal inhibitory concentrations of which were estimated as follows: ampicillin 20 mgL-1, chloramphenicol 10 mgL-1, erythromycin 10 mgL-1, neomycin 20 mgL-1, rifampicin 10 mgL-1 and tetracycline 10 mgL-1.
Figure 2 Scanning electron micrograph of A. phenanthrenivorans strain Sphe3T |
| MIGS ID | Property | Term | Evidence code |
|---|---|---|---|
| Current classification | Domain Bacteria | TAS [7] | |
| Phylum Actinobacteria | TAS [8] | ||
| Class Actinobacteria | TAS [9] | ||
| Subclass Actinobacteridae | TAS [9,10] | ||
| Order Actinomycetales | TAS [9-12] | ||
| Family Micrococcaceae | TAS [9-11,13] | ||
| Genus Arthrobacter | TAS [1,11,14-17] | ||
| Species Arthrobacter phenanthrenivorans | TAS [1] | ||
| Type strain Sphe3 | TAS [1] | ||
| Gram stain | positive | TAS [1] | |
| Cell shape | irregular rods, coccoid | TAS [1] | |
| Motility | Non motile | TAS [1] | |
| Sporulation | nonsporulating | NAS | |
| Temperature range | mesophile | TAS [1] | |
| Optimum temperature | 30°C | TAS [1] | |
| Salinity | normal | TAS [1] | |
| MIGS-22 | Oxygen requirement | aerobic | TAS [1] |
| Carbon source | Phenanthrene, glucose, yeast extract | TAS [1,2] | |
| Energy source | Phenanthrene, glucose, yeast extract | TAS [1,2] | |
| MIGS-6 | Habitat | Soil | TAS [1,2] |
| MIGS-15 | Biotic relationship | Free-living | NAS |
| MIGS-14 | Pathogenicity | none | NAS |
| Biosafety level | 1 | NAS | |
| Isolation | Creosote contaminated soil | TAS [1,2] | |
| MIGS-4 | Geographic location | Perivleptos, Epirus, Greece | TAS [1,2] |
| MIGS-5 | Sample collection time | April 2000 | TAS [1,2] |
| MIGS-4.1 | Latitude | 39.789 | NAS |
| MIGS-4.2 | Longitude | 20.781 | NAS |
| MIGS-4.3 | Depth | 10-20 cm | TAS [1,2] |
| MIGS-4.4 | Altitude | 500 meters | TAS [1,2] |
Evidence codes - IDA: Inferred from Direct Assay (first time in publication); TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from of the Gene Ontology project. If the evidence code is IDA, then the property was directly observed by one of the authors or an expert mentioned in the acknowledgements.
Amylase, catalase and nitrate reductase tests were positive, whereas arginine dihydrolase, gelatinase, lipase, lysine and ornithine decarboxylase, oxidase, urease, citrate assimilation and H2S production tests were negative. No acid was produced in the presence of glucose, lactose and sucrose.
Menaquinones are the sole respiratory lipoquinones of A. phenanthrenivorans strain Sphe3T. Both MK-8 and MK-9(H2) are present in a ratio of 3.6:1, respectively. Major fatty acids are anteiso-C15:0 (36.2%), iso-C16:0 (15.7%), iso-C15:0 (14.3%), anteiso-C17:0 (12.0%), C16:0 (8.3%), iso-C17:0 (4.0%), C16:1ω7c (2.5%) and C14:0 (1.4%). The major phospholipids were diphospatidylglycerol (DPG), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), (63.8, 27.5 and 4.0% respectively).
This organism was selected for sequencing on the basis of its biodegradation capabilities, i.e. metabolizes phenanthrene as a sole source of carbon and energy. The genome project is deposited in the Genome OnLine Database [18] and the complete genome sequence is deposited in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.
| MIGS ID | Property | Term |
|---|---|---|
| MIGS-31 | Finishing quality | Finished |
| MIGS-28 | Libraries used | Three genomic libraries: 6kb (pMCL200) and fosmids (pcc1Fos) Sanger libraries and one 454 pyrosequence standard library |
| MIGS-29 | Sequencing platforms | ABI 3730. 454 GS FLX |
| MIGS-31.2 | Sequencing coverage | 9.33× Sanger, 17.45× pyrosequence |
| MIGS-30 | Assemblers | Newbler version 1.1.02.15, Arachne |
| MIGS-32 | Gene calling method | Prodigal, GenePRIMP |
| INSDC ID | CP002379 | |
| Genbank Date of Release | February 16, 2011 | |
| GOLD ID | Gc01621 | |
| NCBI project ID | 38025 | |
| Database: IMG-GEBA | 2503538005 | |
| MIGS-13 | Source material identifier | DSM 12885 |
| Project relevance | Tree of Life, GEBA |
A. phenanthrenivorans Sphe3T, DSM 18606T was grown aerobically at 30°C on MM M9 containing 0.02% (w/v) phenanthrene. DNA was isolated according to the standard JGI (CA, USA) protocol for Bacterial genomic DNA isolation using CTAB.
The genome of Arthrobacter phenanthrenivorans type strain (Sphe3)was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website [19]. Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 4,967 overlapping fragments of 1,000 bp and entered into assembly as pseudo-reads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and to adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the Arachne assembler [20]. Possible mis-assemblies were corrected and gaps between contigs were closed by by editing in Consed, by custom primer walks from sub-clones or PCR products. A total of 822 Sanger finishing reads were produced to close gaps, to resolve repetitive regions, and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Sanger and 454 sequencing platforms provided 26.78 x coverage of the genome. The final assembly contains 44,113 Sanger reads and 599,557 pyrosequencing reads.
Genes were identified using Prodigal [21] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [22]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and InterPro databases. Additional gene prediction analysis and functional annotation were performed within the Integrated Microbial Genomes - Expert Review (IMG-ER) platform [23].
The genome consists of a 4,250,414 bp long chromosome with a GC content of 66% and two plasmids both with 62% GC content, the larger being 190,450 bp long and the smaller 94,456 bp (Table 3, Figure 3 and Figure 4). Of the 4,288 genes predicted, 4,212 were protein-coding genes, and 76 RNAs; 77 pseudogenes were also identified. The majority of the protein-coding genes (73.8%) were assigned with a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.
| Attribute | Value | % of Total |
|---|---|---|
| Genome size (bp) | 4,535,320 | 100.00% |
| DNA Coding region (bp) | 4,033,112 | 88.93% |
| DNA G+C content (bp) | 2,964,596 | 65.37% |
| Number of replicons | 1 | |
| Extrachromosomal elements | 2 | |
| Total genes | 4,288 | 100.00% |
| RNA genes | 76 | 1.77% |
| rRNA operons | 4 | |
| Protein-coding genes | 4,212 | 98.23% |
| Pseudo genes | 77 | 1.80% |
| Genes with function prediction | 3,167 | 73.86% |
| Genes in paralog clusters | 930 | 21.69% |
| Genes assigned to COGs | 3,075 | 71.71% |
| Genes assigned Pfam domains | 3,277 | 76.42% |
| Genes with signal peptides | 978 | 22.81% |
| Genes with transmembrane helices | 999 | 23.30% |
| CRISPR repeats | 0 |
Figure 3 Graphical circular map of the chromosome, not drawn to scale with plasmids. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew. |
Figure 4 The two plasmids, not drawn to scale with chromosome. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew. |
| Code | value | %age | Description |
|---|---|---|---|
| J | 153 | 4.5 | Translation, ribosomal structure and biogenesis |
| A | 1 | 0.0 | RNA processing and modification |
| K | 308 | 9.0 | Transcription |
| L | 239 | 7.0 | Replication, recombination and repair |
| B | 1 | 0.0 | Chromatin structure and dynamics |
| D | 29 | 0.8 | Cell cycle control, cell division, chromosome partitioning |
| Y | 0 | 0.0 | Nuclear structure |
| V | 45 | 1.3 | Defense mechanisms |
| T | 135 | 3.9 | Signal transduction mechanisms |
| M | 142 | 4.1 | Cell wall/membrane/envelope biogenesis |
| N | 2 | 0.0 | Cell motility |
| Z | 0 | 0.0 | Cytoskeleton |
| W | 0 | 0.0 | Extracellular structures |
| U | 45 | 1.3 | Intracellular trafficking and secretion, and vesicular transport |
| O | 100 | 2.9 | Posttranslational modification, protein turnover, chaperones |
| C | 205 | 6.0 | Energy production and conversion |
| G | 396 | 11.6 | Carbohydrate transport and metabolism |
| E | 329 | 9.6 | Amino acid transport and metabolism |
| F | 87 | 2.5 | Nucleotide transport and metabolism |
| H | 141 | 4.2 | Coenzyme transport and metabolism |
| I | 134 | 3.9 | Lipid transport and metabolism |
| P | 167 | 4.9 | Inorganic ion transport and metabolism |
| Q | 95 | 2.8 | Secondary metabolites biosynthesis, transport and catabolism |
| R | 430 | 12.6 | General function prediction only |
| S | 238 | 6. 9 | Function unknown |
| - | 1,213 | 28.3 | Not in COGs |
This work was supported by the program “Pythagoras II” of EPEAEK with 25% National Funds and 75% European Social Funds (ESF). NCK is supported by the US Department of Energy Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396.
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This work is licensed under a Creative Commons Attribution 3.0 License.
Acknowledgements
We would like to gratefully acknowledge the support of many members of the Genomic Standards Consortium, the broader genomic science community, and those who have indicated their willingness to serve as editors, reviewers and contributors.
Funding for SIGS is provided by a grant from the Office of the Vice President for Research and Graduate Studies at Michigan State University, the Michigan State University Foundation, and the US Department of Energy Biological and Environmental Research DE-FG02-08ER64707.
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